Generating transplantable neurons by in vivo combinatorial screening of transcription regulator RNAs Problem: Neuronal injuries, degenerative diseases, and disorders such as Parkinson's and Huntington's diseases, epilepsy, and stroke affect tens of millions of individuals in the USA alone, and are becoming a more severe problem with the aging population. Although significant effort is being invested for identification of the molecular processes involved, existing chemical and physical therapies do not promise restoration of lost neuronal circuits beyond the short term remedy of symptoms. A potential solution could be the use of tissue transplantation to restore neuronal function. There have been human trials where transplantations, although variably, have resulted in functional recovery in Parkinson's (PD) and Huntington's (HD) diseases. Cell replacement for epilepsy and stroke has shown promise in several rodent studies, and the transplantation of retinal cells to treat degenerative eye diseases is under evaluation in several laboratories. Although there are still many unknowns, in various cases, it has been observed that functional improvements occurred owing to the integration of grafted neurons into existing neuronal networks, and was not simply due to trophic factors released by the transplanted cells. Several studies have also shown that the adult brain is remarkably capable of providing signaling cues that guide the growth of neuronal processes and induce formation of synapses with desired targets when correct cell types are present within the grafts. Xenograft studies with animals larger than rodents have shown that these cues can function over long distances. However, while tissue transplantation may have significant potential, there are many scientific unknowns and several fundamental challenges exist as outlined in this proposal. Handling complexity of these challenges is currently beyond the capabilities of the largest laboratories in the world, and will likely require deployment of systematic high-throughput approaches that will not only address fundamental biological questions and rapidly test various hypotheses without bias, but also provide results that are, if promising, translatable to clinical trials. Challenges: (1) Human fetal or iPS-derived cells are either too scarce or tumorogenic for clinical use, (2) Transplanted cells are too heterogeneous, (3) Preparation of transplanted cells in the correct and synchronized stages is currently impossible, (4) Physical site of transplantation significantly varies from experiment to experiment, (5) Existing in vivo transplantation assays are too slow for screening of multitudes of different hypotheses. Innovation & Methodology: Here, we propose a systematic, unbiased, in vivo, large-scale, and high throughput approach for overcoming these challenges to in vitro differentiation and in vivo testing of transplanted neuronal tissues. The proposed methodologies here are applicable to most transplantation paradigms. The key technologies and strategies we will develop include: (A) RNA-mediated nuclear reprogramming without genetic modification, (B) Reprogramming human cell lineages by systematic ultra-high-throughput screening of RNA transcription factor cocktails using a massively parallel technology, (C) High-throughput transplantation of human cells into rodents (with minimal rodent sacrifice) and in vivo analysis of neuronal survival and integration.